Probing the Sources of the Apparent Irreproducibility of Amyloid Formation: Drastic Changes in Kinetics and a Switch in Mechanism Due to Micellelike Oligomer Formation at Critical Concentrations of IAPP

Abstract

The aggregation of amyloidogenic proteins is infamous for being highly chaotic, with small variations in conditions sometimes leading to large changes in aggregation rates. Using the amyloidogenic protein IAPP (islet amyloid polypeptide protein, also known as amylin) as an example, we show that a part of this phenomenon may be related to the formation of micellelike oligomers at specific critical concentrations and temperatures. We show that pyrene fluorescence can sensitively detect micellelike oligomer formation by IAPP and discriminate between micellelike oligomers from fibers and monomers, making pyrene one of the few chemical probes specific to a prefibrillar oligomer. We further show that oligomers of this type reversibly form at critical concentrations in the low micromolar range and at specific critical temperatures. Micellelike oligomer formation has several consequences for amyloid formation by IAPP. First, the kinetics of fiber formation increase substantially as the critical concentration is approached but are nearly independent of concentration below it, suggesting a direct role for the oligomers in fiber formation. Second, the critical concentration is strongly correlated with the propensity to form amyloid: higher critical concentrations are observed for both IAPP variants with lower amyloidogenicity and for native IAPP at acidic pH in which aggregation is greatly slowed. Furthermore, using the DEST NMR technique, we show that the pathway of amyloid formation switches as the critical point is approached, with self-interactions primarily near the N-terminus below the critical temperature and near the central region above the critical temperature, reconciling two apparently conflicting views of the initiation of IAPP aggregation.

What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

“…2c). Characteristic but not exclusive of HEWL aggregation, the low reproducibility of the results further prevents definitive conclusions to be made without testing wider ranges of protein concentrations and numerous other replicates (40,43,47,48). The previously reported formation of intermediate and off-pathway species during amyloid fibrillization of lysozyme should, however, produce atypical kinetic signatures and explain in part the poor reproducibility indexes.…”

“…A striking result not covered by any of the scenarios in Fig. 1b is the weak dependence of the lag time on concentration with absolute values of ␥ well below 1 at high monomer concentrations (15,31,40,43). In the case of lysozyme aggregation under harsh denaturating conditions, the process becomes nearly concentration-independent (␥ Ӎ 0) for protein concentrations significantly higher than the solubility (40).…”

Amylin–Aβ oligomers at atomic resolution using molecular dynamics simulations: a link between Type 2 diabetes and Alzheimer's disease

“…60 Previous ssNMR studies have shown α-helical structures of Amylin and not cross-β structure. 61,62 Recent ssNMR studies presented unstructured Aβ oligomers 63 and Amylin oligomers that form large micelles, 64 which may be a general phenomenon for natively unstructured Amylin. We did not apply these α-helical and the unstructured amylin, or the unstructured Aβ oligomers in the current study, because of the lack of the PDB coordinates.…”

Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy

“…Human-IAPP forms “micelle-like” mesoscopic aggregates whereas the rat-IAPP does not [90]. It is also reported that micelle formation correlated with amyloidogenicity of the peptide: the naturally-occurring C-amidated human-IAPP exhibited about 10 times less CMC than the non-amidated while the human-IAPP-1–19 fragments of both human and rat did not form micelles [109]. Studies have shown that the presence of lipid influences the aggregation of IAPP [86,93–98,117–125].…”